Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia

Radiotherapy in Combination With Cytokine Treatment

Radiotherapy (RT) plays an important role in the management of cancer patients. RT is used in more than 50% of patients during the course of their disease in a curative or palliative setting. In the past decades it became apparent that the abscopal effect induced by RT might be dependent on the activation of immune system, and that the induction of immunogenic cancer cell death and production of danger-associated molecular patterns from dying cells play a major role in the radiotherapy-mediated anti-tumor efficacy. Therefore, the combination of RT and immunotherapy is of a particular interest that is reflected in designing clinical trials to treat patients with various malignancies. The use of cytokines as immunoadjuvants in combination with RT has been explored over the last decades as one of the immunotherapeutic combinations to enhance the clinical response to anti-cancer treatment. Here we review mainly the data on the efficacy of IFN-α, IL-2, IL-2-based immunocytokines, GM-CSF, and TNF-α used in combinations with various radiotherapeutic techniques in clinical trials. Moreover, we discuss the potential of IL-15 and its analogs and IL-12 cytokines in combination with RT based on the efficacy in preclinical mouse tumor models

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC− toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca2+ influx promoted by molecules locked in a Ca2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux

Payload diversification: a key step in the development of antibody–drug conjugates

Abstract Antibody–drug conjugates (ADCs) is a fast moving class of targeted biotherapeutics that currently combines the selectivity of monoclonal antibodies with the potency of a payload consisting of cytotoxic agents. For many years microtubule targeting and DNA-intercalating agents were at the forefront of ADC development. The recent approval and clinical success of trastuzumab deruxtecan (Enhertu®) and sacituzumab govitecan (Trodelvy®), two topoisomerase 1 inhibitor-based ADCs, has shown the potential of conjugating unconventional payloads with differentiated mechanisms of action. Among future developments in the ADC field, payload diversification is expected to play a key role as illustrated by a growing number of preclinical and clinical stage unconventional payload-conjugated ADCs. This review presents a comprehensive overview of validated, forgotten and newly developed payloads with different mechanisms of action

Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy

<div><p>High hydrostatic pressure (HHP) induces immunogenic death of tumor cells which confer protective anti-tumor immunity <i>in vivo</i>. Moreover, DC pulsed with HHP-treated tumor cells induced therapeutic effect in mouse cancer model. In this study, we tested the immunogenicity, stability and T cell stimulatory activity of human monocyte-derived dendritic cell (DC)-based HHP lung cancer vaccine generated in GMP compliant serum free medium using HHP 250 MPa. DC pulsed with HHP-killed lung cancer cells and poly(I:C) enhanced DC maturation, chemotactic migration and production of pro-inflammatory cytokines after 24h. Moreover, DC-based HHP lung cancer vaccine showed functional plasticity after transfer into serum-containing media and stimulation with LPS or CD40L after additional 24h. LPS and CD40L stimulation further differentially enhanced the expression of costimulatory molecules and production of IL-12p70. DC-based HHP lung cancer vaccine decreased the number of CD4⁺CD25⁺Foxp3⁺ T regulatory cells and stimulated IFN-γ-producing tumor antigen-specific CD4⁺ and CD8⁺ T cells from non-small cell lung cancer (NSCLC) patients. Tumor antigen specific CD8⁺ and CD4⁺ T cell responses were detected in NSCLC patient’s against a selected tumor antigens expressed by lung cancer cell lines used for the vaccine generation. We also showed for the first time that protein antigen from HHP-killed lung cancer cells is processed and presented by DC to CD8⁺ T cells. Our results represent important preclinical data for ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa) in combination with chemotherapy and immune enhancers.</p></div

DC-based HHP lung cancer vaccine displays mature phenotype, produces pro-inflammatory cytokines and increases chemotactic migration.

<p>2 × 10⁵ monocyte-derived DC generated in serum-free media X-VIVO 15 were left untreated [iDC] pulsed with thawed 4 × 10⁴ HHP-killed lung cancer cells [HHP], pulsed with HHP-killed cells and poly(I:C) [HHP+poly(I:C)] or stimulated with poly(I:C) only [poly(I:C)] for 24h. (A) The expression of CD80, CD86, CD83, HLA-DR and (E) CCR7 on CD11c⁺DAPI negative cells was assessed by flow cytometry. The values represent means ± SEM of 16 donors. Histograms are representative of 16 donors. (B) For phagocytic assays thawed HHP-killed H520 and H522 cells were stained with VybrantVR DiD and DC were stained with VybrantVR DiO at 37°C for 20 min before vaccine generation. The phagocytic capacity of DC was assessed as a percentage of DiO⁺DiD⁺ cells by flow cytometry after 24h. Graphs represent means ± SEM of n = 3 in duplicates. (C) Cytokine production was evaluated in cell culture supernatants after 24h by Luminex assay. Graphs represent means ± SEM of n = 5. (D) 3 × 10⁵ DC treated as described above were transferred to serum-containing media in upper chamber of transwell plate and were allowed to migrate to the lower chamber filled with media or media containing a mixture of CCL19 and CCL21 (both 50 ng/ml) at 37°C for 5h. The number of transmigrated CD11c⁺ DC was determined by flow cytometry. The graph shows mean ± SEM of 16 donors of n = 4. The results were considered statistically significant if * p < 0.05, ** p < 0.01 or *** p < 0.001.</p

DC-based HHP lung cancer vaccine generated from NSCLC patients’ monocytes induces tumor-antigen specific CD8⁺ and CD4⁺ T cells.

<p>4 × 10⁴ iDC, DC pulsed with thawed HHP-killed lung cancer cells or MP1-expressing A549 and poly(I:C) [HHP+poly(I:C)] or stimulated with poly(I:C) alone [poly(I:C)] for 24h were subsequently added to 2 × 10⁵ autologous T cells from NSCLC patients and subsequently co-cultured for 7 days. (A) The MP1_58-66-specific CD8⁺ T cells were detected without restimulation with MP1_58-66-HLA-A*201 Tetramer staining by flow cytometry. Dotplots are representative of 4 donors. Graphs represent means ± SEM of 4 donors from n = 2. (B) IFN-γ-producing CD8⁺ and CD4⁺ T cells were assessed by flow cytometry after one round of restimulation with freshly prepared NSCLC patient’ DC. The percentage in graphs is displayed as a percentage of IFN-γ-producing cells from CD8⁺ or CD4⁺ T cells, respectively, therefore it does not correspond to percentages in dotplots. (C) T cell proliferation (Ki-67) was determined without restimulation by flow cytometry. Dotplots are representative of 6 patients. Graphs show means of ± SEM of 6 donors in duplicates. The results were considered statistically significant if * p < 0.05, ** p < 0.01 or *** p < 0.001. (D) qPCR analyses of Mucin-1, hTERT, Survivin, MAGE-A3 and MAGE-A4 expression in H520 and H522 cell lines (n = 3, in duplicates) (E) Gating strategy to detect tumor antigen specific IFN-γ producing T cells in NSCLC patients. One representative patient sample for MAGE-A4-specific T cells is shown. (F) Quantitative evaluation of tumor antigen specific IFN-γ producing T cells in 36 NLCLC patients. The percentage of negative control (no antigen) was deducted from the samples stimulated with antigenic pepmixes.</p

DC-based HHP lung cancer vaccine stimulates CD8⁺ and CD4⁺ T cells and decreases the number of CD4⁺CD25⁺Foxp3⁺T regulatory cells.

<p>4 × 10⁴ iDC, DC pulsed with thawed HHP-killed lung cancer cells and poly(I:C) [HHP+poly(I:C)], or stimulated with poly(I:C) alone [poly(I:C)] for 24h were subsequently added to 2 ×10⁵ autologous T cells from healthy donors and subsequently co-cultured for 7 days. (A) IFN-γ-producing CD8⁺ and CD4⁺ T cells were assessed by flow cytometry after one round of restimulation with freshly prepared DC. CD8⁺ T cells shown in dotplots are gated from CD3⁺ cells, and predominantly CD4⁺ T cells are displayed as CD8 negative cells. The percentage in graphs is displayed as a percentage of IFN-γ-producing cells from CD8⁺ or CD4⁺ T cells, respectively, therefore it does not correspond to percentages in dotplots. (B) T cell proliferation (Ki-67) and (C) the number of CD4⁺CD25⁺FoxP3⁺T regulatory cells was determined without restimulation by flow cytometry. Dotplots are representative of 8 donors. Graphs represent means ± SEM of 8 donors from n = 3. The results were considered statistically significant if * p < 0.05, ** p < 0.01 or *** p < 0.001.</p

DC-based HHP lung cancer vaccine further increases its maturation and cytokine production in serum containing medium after additional LPS and CD40L stimulation.

<p>2 × 10⁵ monocyte-derived DC generated in serum-free media X-VIVO 15 were left untreated [iDC] pulsed with thawed 4 × 10⁴ HHP-killed lung cancer cells and poly(I:C) [HHP+poly(I:C)] or stimulated with poly(I:C) only [poly(I:C)] for 24h [24h]. Serum-free medium was subsequently replaced with medium containing 10% human AB serum alone or supplemented with LPS (1 μg/ml) (A) or cells were loaded onto a layer of CD40L-expressiong A549 (B) for additional 24h [48h]. Non-transfected A549 cells were used as a negative control of CD40L stimulation (B). The difference in CD80, CD86, CD83 and HLA-DR expression between 24h and 48h samples was determined by flow cytometry. Similarly, the difference in IL-12p70 and IL-10 production was assessed by ELISA. Graphs show mean ± SEM of 8–18 donors of n = 5. The results were considered statistically significant if * p < 0.05, ** p < 0.01 or *** p < 0.001.</p